Ovarian cancer is one of the ten most common types of cancer in women and an estimated 1 in 71 women in the United States will develop ovarian cancer in their lifetime. Regrettably, almost 75 % of women are diagnosed with advanced disease, which greatly increases mortality. Hence, earlier and accurate characterization of adnexal masses is a clinical imperative in order to improve patient's survival and imaging techniques can play a critical role in fulfilling this goal. Currently adnexal masses are imaged with standard endovaginal ultrasound (EV US), with contrast enhanced magnetic resonance imaging (MRI) or computed tomography (CT), but functional biomarkers may be important prognostic parameters to improve on the characterization of such masses. Our group has been developing a new contrast-specific US imaging modality: subharmonic imaging (SHI), because of the excellent suppression of tissue echoes relative to other US contrast modes. We have shown that SHI can detect the slow, small volume blood flow associated with tumor angiogenesis in humans using clinically approved dosages of US contrast agents. Recently, we created parametric images of SHI kinetics (i.e., pixel by pixel time intensity curves; TICs) and found cancers to have significantly higher perfusion relative to benign lesions (p = 0.0014). Hence, the fundamental aim of this project is to derive qualitative and quantitative SHI biomarkers, based on subharmonic signals from US contrast microbubbles, which can be used to improve the characterization of adnexal masses as benign or malignant. The SHI algorithm will be implemented on a state-of-the-art US scanner (EPIQ; Philips Healthcare, Bothell, WA) with a broad bandwidth, curved array EV transducer for in vivo SHI of adnexal masses (Specific Aim 1). The ovaries of 6 canines will be imaged to demonstrate the ability of SHI to depict macro- and micro-vascularity (the latter as a model for tumor angiogenesis). Next, perfusion in the ovaries will be estimated using SHI TICs and results compared to a neutron activation assay. As a proof-of-concept, pilot study, we will enroll approximately 45 women with adnexal masses scheduled for surgery based on the risk of malignancy index (RMI). Subjects will be studied with EV SHI to identify and optimize TIC parameters as SHI biomarkers to evaluate adnexal microvascularity in an attempt to diagnose benign or malignant disease. The TIC parameters will also be converted into SHI parametric images and compared to the standard EV US as well as MRI and RMI lesion characterization with pathology as the reference standard (Specific Aim 2). Finally, the ability of EV SHI to image angiogenesis will be compared to surgical specimens stained for CD31, an immunohistochemical predictor of angiogenesis (Specific Aim 3). This will be a first step towards the long-term goal of translating this method into a clinical trial for screening of high-risk patients with adnexal masses.